Browsing by Subject "Gluconeogenesis"
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Item Carbon Starvation Metabolically Regulates Chromatin for Transcriptome Rewiring(2022-05) Hsieh, Wen-Chuan; Conrad, Nicholas; Kraus, W. Lee; Orth, Kim; Tu, BenjaminCells robustly rewire their transcriptomes to survive under stress conditions. Yet, how does such reprogramming of gene expression occur? Under favorable nutrient conditions, acetyl-CoA normally promotes histone acetylation to activate genes required for cell growth. However, glucose starvation significantly reduces the availability of acetyl-CoA. And it is unclear how such a change impacts genome-wide histone acetylation and gene expression. In this study, I set up a robust glucose starvation model in budding yeast to discover a mechanism by which cells preserve acetyl-CoA, a key intermediate in energy metabolism, in order to sustain histone acetylation for gene activation even under stress conditions. I demonstrate a dramatic redistribution of histone acetylation upon glucose starvation. Mechanistically, I determined that a major histone deacetylase (HDAC) releases acetyl groups from histones at growth-promoting genes, which can subsequently be used to acetylate histones at a distinctive set of stress-responsive genes. Strikingly, bioinformatic analysis revealed these genes to be required for gluconeogenic and fat metabolism, which are metabolic pathways that generate acetyl-CoA for oxidation and ATP synthesis. Genetic deletion of histone modifiers mediating this reallocation, including the key HDAC or histone acetyltransferase (HAT), disrupts proper transcriptome rewiring for survival. Given the importance of acetate for recycling the acetyl- group, I next characterize acetyl-CoA synthetases (Acs), metabolic enzymes that convert acetate to acetyl-CoA. I demonstrate that Acs2 is required for maintaining global histone acetylation, yet its nuclear localization appears to be dispensable for such regulation. I observe that the catalytic activity of Acs2 governs the intracellular acetyl-CoA level and global histone acetylation amounts. Compromising its activity leads to up-regulation of ergosterol biosynthetic pathways in addition to gluconeogenic and fat metabolism genes upon glucose starvation. In summary, I reveal an unexpected switch in the specificity of histone acetylation to promote pathways that generate acetyl-CoA for oxidation when acetyl-CoA is limiting. I have elucidated how transcriptome rewiring is driven by reallocation of histone acetylation. My findings present a mechanism by which cells recycle acetyl groups to differentially acetylate histones for activation of key genes required for metabolism and survival.Item Effects of SGLT-2 Inhibitors and Visceral Fat on Glucose Metabolism(2018-01-23) Hughes, Connor; Bleiberg, Ben; Ayers, Colby R.; Mallory, Craig R.; Jin, Eunsook S.; Neeland, Ian J.INTRODUCTION: Abdominal obesity and excess visceral adiposity (VAT), have strong associations with insulin resistance, hyperglycemia and type 2 diabetes. A previous pilot study showed that participants with high VAT have less 13C enrichment in glucose reflecting an abundant endogenous substrate pool from adipose turnover for gluconeogenesis, compared with participants with low VAT. This study aims to evaluate the effects of a SGLT-2 inhibitor known to modify markers of VAT on gluconeogenic pathways in the liver. METHODS: Obese adults without diabetes were stratified into high and low VAT groups based on MRI (high n=8, low n=7). After an overnight fast, participants were administered non-radioactive labeled glycerol, and blood samples were collected to calculate glycerol enrichment at sequential time points over 180 minutes. Participants were then randomized to receive empagliflozin 10 mg daily or matching placebo for 3 months and glycerol studies were repeated. RESULTS: High VAT subjects demonstrated a significantly lower enrichment of ingested glycerol in blood glucose, when compared to low VAT individuals for the 60-180 minute interval (p <0.001). During this same time interval, pentose phosphate pathway activity was significantly decreased in high VAT, compared to low VAT subjects (p<0.01). The effects of empagliflozin on these pathways are currently being analyzed. DISCUSSION: SGLT-2 inhibitors inhibit renal glucose reabsorption in the proximal nephron, inducing weight loss and decreasing systolic blood pressure. This medication has been suggested to reduce CVD event rate and lower hemoglobinA1c levels up to 1%. Observed differences in pathways between high visceral fat subjects on a SGLT-2 inhibitor compared to control high visceral fat subjects could provide insight into the physiologic changes provided by SGLT-2 inhibitors.Item Effects of Visceral Adiposity on Glycerol Pathways in Gluconeogenesis(2017-01-17) Hughes, Connor; Neeland, Ian J.; Ayers, Colby R.; Malloy, Craig R.; Jin, Eunsook S.OBJECTIVE: To determine effects of visceral adiposity on multiple pathways in gluconeogenesis from glycerol in obese humans. RESEARCH DESIGN AND METHODS: Obese (BMI ≥30 kg/m2) participants without type 2 diabetes underwent visceral adipose tissue (VAT) assessment and were stratified by median VAT into high VAT-fasting (n=3), low VAT-fasting (n=4), and high VAT-refed (n=2) groups. Participants ingested [U-13C3]glycerol and blood samples were subsequently analyzed at multiple time points over 3 hours by NMR spectroscopy. The fractions of plasma glucose (enrichment) derived from [U-13C3] glycerol via hepatic gluconeogenesis, pentose phosphate pathway (PPP), and tricarboxylic acid (TCA) cycle were assessed using 13C NMR analysis of glucose. Mixed linear models were used to compare 13C enrichment in glucose between groups. RESULTS: Mean age, BMI, and baseline glucose was 49 years, 40.1 kg/m2, and 98 mg/dl, respectively. Up to 20% of glycerol was metabolized in the TCA cycle prior to gluconeogenesis and PPP activity was minor (<1%) in all participants. There was a 21% decrease in 13C enrichment in plasma glucose in the high VAT-fasting compared with low VAT-fasting group (p=0.03), suggesting dilution by endogenous glycerol. High VAT-refed participants had 37% less 13C enrichment in glucose compared with high VAT-fasting (p=0.02). There was a trend toward lower [1,2-13C2] (via PPP) and [5,6-13C2] (via TCA cycle) glucose in high VAT versus low VAT groups. CONCLUSIONS: We applied a simple method to detect gluconeogenesis from glycerol in obese humans. Our findings provide preliminary evidence that excess visceral fat disrupts multiple pathways in hepatic gluconeogenesis from glycerol.Item Non-Canonical Roles of Cytosolic Phosphoenolpyruvate Carboxykinase in Small Intestine Metabolism(2016-03-30) Potts, Austin Joseph; Repa, Joyce J.; Burgess, Shawn C.; Tu, Benjamin; Browning, JeffreyPhosphoenolpyruvate carboxykinase (PEPCK) is considered by many to be the quintessential regulatory enzyme of gluconeogenesis. This common perspective of PEPCK solely functioning in gluconeogenesis is too narrow and does not do justice to the complexity and number of pathways in which this enzyme participates. PEPCK performs important roles in a number of tissues: kidney, for acid-base balance with concomitant gluconeogenesis; adipose, with glyceroneogenesis for fatty acid esterification; enterocytes of the small intestine, for amino acid oxidation; and most famously, gluconeogenesis in the liver. Though amino acid, fatty acid, and glucose metabolism of the small intestine have been well studied, no investigation has emphasized the role of intestinal PEPCK in these pathways. The aim of this work is to examine the roles of PEPCK in small intestine function and metabolism. A mouse model with an intestine-specific deletion of PEPCK was generated by crossing existing models to produce Pck1ff and Pck1ff+Villin-cre+/-, named Small Intestine PEPCK Knockout (SIPKO). Intestinal metabolism was evaluated in vivo and in vitro with respect to glucose, triglycerides, amino acids, and TCA cycle metabolism using a novel everted intestine perfusion model, isolated enterocytes, 13C stable isotope tracers, GC-MS, LC-MSMS, 13C NMR spectroscopy, and mathematical modeling. The functions of PEPCK in small intestine enterocytes largely recapitulate known roles in other tissues. Although, intestinal gluconeogenesis was ablated in the SIPKO mouse, there was no change in whole body glucose homeostasis. However, postprandial triglyceride secretion was attenuated and intestinal amino acid profiles were dramatically different. Nearly all non-essential amino acid concentrations were increased, and nearly all essential amino acid concentrations were decreased. Intestinal TCA cycle and related pathway fluxes were decreased by approximately half, and enterocyte oxygen consumption was lower during a TCA cycle challenge. These studies indicate that the metabolic roles of PEPCK in the small intestine extend far beyond gluconeogenesis to include cataplerotic functions that not only include glucose production, but also triglyceride storage, amino acid catabolism and oxidative energy production. In summary, our studies reveal intestinal PEPCK is a metabolically influential enzyme that is at the hub of macronutrient metabolism and facilitates TCA cycle flexibility and adaptability.Item [Southwestern News](2001-09-17) Wren, Worth, Jr.